The Tracking Study In The Tibet Miniature Pig Of Bone Marrow Stromal Cells Labeled With Superparamagnetic Iron Oxide In Vivo

PartⅠNew Superparamagnetic iron oxide particles efficiently labeled BMSCs without injuring valiability in vitroObjective: To evaluate the safety and efficiency of Tibet miniature pig bone mesenchymal stem cells (BMSCs) labeled by new superparamagnetic iron oxide (SPIO) nanoparticles, and assess characteristics of magnetic resonance imaging(MRI) in vitro.Methods BMSCs were isolated from bone marrowb aspirates of the Tibet miniature pig by density gradient centrifugration with Ficoll lymphocyte separation medium, and cultured, expanded, identifyed, after which, they were incubated with various concentrations of SPIO particles. The BMSCs labeling efficiency was tested with prussian blue staining. Intracytoplasmic iron particles was shown by the transmission electron microscopy . The growth ability, proliferation, viability and cell cycle was tested with MTT, Trypan blue and Propidium Iodide respectively. The differentiation of labeled cells to adipogenic and osteogenic cells was induced with induction medium. An optimal concentration of SPIO and minimum number of BMSCs imaged with MR was detected with T1WI and T2*WI and measured signal intensity by laddering concentration(12.5,25,50ug/mL) and cell number(1×10~6, 1×10~5 ,1×10~4 ,1×10~3 , 1×10~2),that samples of SPIO- labeled and unlabeled MSCs was floated in agar.Result: Nearly 100% of the BMSCs were labeled with SPIO by Prussian blue staining. Intracytoplasmic iron nanoparticles were observed by transmission electron microscopy. SPIO at 25ug/mL concentration and treatment times of 24 h did not statistically affect the viability,proliferation,cell cycle and differentiation potential of BMSCs and shown best in MR, the number 1×10~3 can be visible with MR. SPIO labelingcaused a stronger low signal attenuation effect in GRE(T2*WI).Conclusion: In vitro, BMSCs can be easily and efficiently labeled with new SPIO, and SPIO at 25ug/mL concentration may be a optimal imaging without interference on the cell viability and proliferation, MRI visualization of SPIO-labeled BMSCs is feasible in T2*WI.PartⅡTracing study of SPIO-labeled BMSCs in physiological Tibet miniature pig in vivoObjective To observe distribution of SPIO-labeled BMSCs with 25ug/mL concentration by the different ways infusion in physiological condition of Tibet miniature pig.Methods: BMSCs of Tibet miniature pig were labeled with SPIO. Tibet miniature pig were injected (2×10~6/Kg) SPIO-labeled BMSCs through peripheral venous and renal arterial .One hour afer infusion , Tibet miniature pig were imaged with MR. One day later, the pathological specimens of the heart, liver, spleen, lung, pancreas and both kidneys were taken. Prussian blue straining and H-E straining were performed with the tissue slices to find out SPIO-labeled BMSCs and observe its distribution in various organs.Results: Signal intensity loss in the kidney of Tibet miniature pig was not observe on the T2*WI sequence which persisted 1 hour after SPIO-labeled BMSCs were injected by peripheral venous and renal arterial, that is meaning MR can not distinguish this concentration(25ug/mL) BMSCs infection. Histological analysis by prussian blue straining confirmed SPIO-labeled BMSCs were capyured by the lung after infusion through peripheral venous, and the cells were found in the infused kidney and in the lung after infusion by renal arterial .Conclusion: In physiological condition of Tibet miniature pig, SPIO-labeled BMSCs were captured by the lung after infusion through peripheral venous, and the cells were found in the infused kidney and in the lung after infusion by renal arterial.PartⅢTracing study of SPIO-labeled BMSCs in transplanted renal allograft Tibet miniature pig in vivoObjective To observe distribution of SPIO-labeled BMSCs by the different ways infusion in Tibet miniature pig after renal transplantation, and find out differentiation of BMSCs distribution by infused in peripheral venous and transplanted renal arterial respectively.Methods: To establish renal transplant models of Tibet miniature pig recipients. The recipients were injected (2×10~6/Kg) SPIO-labeled BMSCs through peripheral venous and renal arterial of the graft after renal transplantation. One day afer infusion, the histological specimens of the heart, liver, spleen, lung, pancreas, native kidney and transplanted kidney were taken. Prussian blue straining and H-E straining were performed with the tissue slices to find out SPIO-labeled BMSCs and observe its distribution in various organs.Results: Histological analysis by prussian blue straining confirmed that SPIO-labeled BMSCs were only found in the lung after infusion through peripheral venous, but cells were discovered in the lung and in the transplanted kidney also after infusion by transplanted renal arterial ,and most of SPIO-labeled BMSCs located in the glomeruli of kidney cortex, and few cells situated in the lung interstitial. SPIO-labeled BMSCs were statistically more in the lung by peripheral venous compared to by transplant renal artery (P <0.05), at same number of BMSCs were infused.Conclusion: After kidney transplantation, SPIO-labeled BMSCs were captured in the lung by peripheral venous, and were found in the lung and infused kidney after infusion by transplanted renal arterial, there are number differences of BMSCs in the lung tissue .